Historically, man has manipulated the genetic structure of microorganisms, plants and animals primarily by selection of desirable natural mutants of living organisms or by cross fertilization of organisms followed by selection of a desirable strain. Such methods have given us desirable microorganisms such as the yeasts that are used in baking and that are used in fermentation for the manufacture of beverages such as beer and wine. Other such microorganisms produce antibiotics and others are responsible for production of certain foods such as pickles and sauerkraut. Other selected microorganisms are used in desirable degradation processes such as the microorganisms used in waste disposal. Such genetic manipulation has not been limited to microorganisms and has also resulted in improved species of plants and animals such as hybrid food crops and animals having desirable meat, milk or egg production.
It has recently become technically possible to move genes from one cell type to another (usually from plants and animals to bacteria) by use of techniques developed in the study of molecular biology of prokaryotic cells (cells which are prenucleus) and eukaryotic cells (cells which contain a nucleus which are usually cells of higher organisms). Such a result is exceedingly desirable since man no longer has to rely on the appearance of spontaneous mutants. It is now possible to transfer genes from higher plants or animals (eukaryotic cells) to place them into bacterial cells (prokaryotic cells) by means of a vector. "Genes", as used herein, means a segment of DNA (deoxyribonucleic acid) which carries genetic information. "Vector", as used herein, is any composition or structure which can carry genes into the cell for replication (manufacture of additional similar DNA fragments), and also usually for transcription (manufacture of an RNA segment) and translation (manufacture of a polypeptide, usually a protein, from the information contained in an RNA segment). The vector is usually a phage virus to which the gene has been attached or a plasmid (circular rings of of DNA which are relatively small in size when compared with the length of chromosomal DNA). Chromosomal DNA is a long string of DNA which contains most of the genetic information in a cell.
The structure of DNA and RNA (ribonucleic acid) is based upon the arrangement of bases along alternating residues of certain sugars and phosphate. In the case of DNA, the alternating sugar residue is deoxyribose and in the case of RNA, the alternating sugar residue is ribose. The bases in the case of DNA are radicals of the chemicals thymine, cytosine, adenine, or guanine. In RNA, the bases are uracil, cytosine, adenine, or guanine. It is the arrangement of the bases which determines the genetic information. RNA is usually either messenger RNA (mRNA) which carries information from the DNA as an intermediary in the formation of polypeptides or is a transfer RNA (tRNA) which acts between the messenger RNA and amino acids to combine the amino acids in a particular sequence based upon the sequence and information contained in the messenger RNA. The transfer RNA seems to attach to both the information RNA and to a particular amino acid thus arranging the amino acids in the proper order. Each amino acid has its own transfer RNA which recognizes only particular sequence along a messenger RNA thus making certain that the old sequence in the messenger RNA is properly translated into the appropriate amino acid. Sequences of amino acids (polypeptides), usually a protein, may have many different functions depending upon the particular sequence. Such polypeptides may for example act as enzymes which are organic catalysts, hormones which act as regulators, antibodies which are produced in response and defense against foreign materials called antigens, structural and contractile proteins and blood and plasma proteins including albumins, fibrinogen vital in blood clotting and hemoglobin which carries oxygen.
It has been known that bacterial plasmids such as those found in the bacterial genuses salmonella, shigella, proteus, bacillus, pseudomonoas, streptomyces and all gram negative enteric bacteria such as Escherichia coli could be cleaved, new genetic codes (usually for a desired polypeptide) could be then inserted into the plasmid and the plasmid could then be replaced into a bacteria for replication generally followed by transcription and translation to form the desired polypeptide structure. "Cleaved", as used herein, is intended to mean cleaved or broken. "Restricted", is intended to mean cleaved by any means but usually by use of a restriction enzyme.
It has been further recognized that one method for cleaving is by utilizing restriction enzymes followed by insertion of the desired DNA sequence. In particular, such methods are set forth in "Molecular Cloning A Laboratory Manual" by Maniatis et al, published 1982 by Cold Spring Harbor Laboratory. Table 4.1 beginning on page 100 of the manual lists numerous restriction enzymes and the sequence and location of cleavage.
Although such procedures are well known to those skilled in the art, there remain serious problems with respect to the utilization of inserted plasmids for replication of the plasmid, transcription of the inserted sequence to form the appropriate RNA and translation of messenger RNA to the appropriate polypeptide.
In the prior art, it was recognized that methods had to be used to maintain the desired plasmid within the bacterial organism. One effective procedure was to include a gene for antibiotic resistance in the plasmid and then grow the bacteria in a media containing the particular antibiotic. The result was that only bacteria which contained the plasmid could continue to grow since bacteria without the plasmid were unable to cope with the hostile environment. Such a method, however, required that antibiotics be added to the growth medium, which in commercial production was an undesirable, costly and inconvenient step. Furthermore, there was a tendency of many microorganisms to develop the ability to resist antibiotics by a mutation on the chromosome which then made the plasmid unnecessary within the cell.
It was also known in the prior art that bacteria having a gene for an enzyme to make an essential protein, which gene was defective due to the presence of a nonsense codon, could function when a plasmid, having a gene for an abnormal tRNA which could read the nonsense codon and insert a suitable amino acid, was present in the cell. In the absence of the plasmid, the bacteria would die since the essential protein would no longer be produced.
This method was somewhat effective; however, there was a tendency for the mutated gene for the enzyme to revert back to a normal gene and there was also a tendency for the chromosomal DNA to develop its own code for a tRNA which could read the nonsense codon, both of which made the plasmid unnecessary to the cell.
In the prior art, it was recognized that ribosomal RNA operators were strong operators but there was extreme difficulty in making the operators function outside of their natural location. It has recently been found that an operon containing ribosomal RNA operators could be made to function provided that the parts of both the beginning and end of the operon was utilized, i.e. especially the promoter and promoter termination sequence. The "promoter termination sequence" is the termination sequence properly associated with termination of transcription commencing at the promoter. The promoter termination sequence is in general the sequence which is desired for termination of transcription commencing at the promoter rather than premature termination which may be caused by undesirable nonsense codons or premature, usually undesirable, sequences which act as terminators located between the promoter and the promoter termination sequence. The promoter termination sequence has also been termed the "transcription termination sequence".
A complete ribosomal RNA operon, including the natural sequence between the promoter and promoter termination sequence, was inserted in the prior art into a plasmid (see Morgan et al "Some rRNA Operons in E. coli have tRNA Genes at their Distal Ends". Cell, Volume 13, pages 335-344 1978). Such a plasmid had little utility since the plasmid was exceedingly large, i.e., about 27,000 base pairs long and had an exceedingly large number of base pairs in the ribosomal RNA operon including operators, promoter, promoter termination sequence, and intermediate sequence (the sequence between the ribosomal RNA promoter and promoter termination sequence). The entire operon had a size of about 5,800 base pairs.
The large size of the plasmid made it unsuitable for genetic engineering purposes since plasmids of such large size are generally rapidly lost from a bacterial cell and since plasmids of such large size replicate slowly. In addition, the long operon made cleavage of the intermediate sequence, followed by insertion of a new desired DNA sequence, impractical. Such impracticality partially results due to the large number of restriction nuclease sites in such a long intermediate sequence and in such a large plasmid. Furthermore, such sites and their location are difficult to characterize. In addition, the insertion of an additional sequence into such a large plasmid would make the plasmid even more unstable in the bacterial organism.
In the prior art, it was also recognized that RNA's could be analyzed by gel electrophoresis. C. Ikemura et al "Small Ribonucleic Acids of Escherichia coli: 1. Characterization by Polyacrylamide Gel Electrophoresis and Fingerprint Analysis", Journal of Biological Chemistry, Vol 248, pp. 5024-5032 and Peacock, et al "Resolution of Multiple Ribonucleic Acid Species by Polyacrylamide gel Electrophoresis" Biochemistry Vol 6, pp. 1818-1827. It was also recognized that certain tRNA's from plasmids could be analyzed by gel electrophoresis as discussed by Ikemura et al "Expression of Spacer tRNA genes in Ribosomal RNA Transcription Units carried by Hybrid ColE1 Plasmids in E.coli", Cell, Vol 11, pp. 779-793. Such test procedures, eg. for analyzing for transfer RNA's, had never been used in a small plasmid which rapidly replicates to test for transcription of a gene upstream of the transfer RNA sequence.